A major impediment to worldwide vaccination efforts is the thermal lability of vaccines. Most vaccines are currently stored at temperatures below ambient through the maintenance of a “cold chain” from the manufacturing site to the administration site. While improvements in cold chain maintenance have been made, this system is expensive, remains error prone, and it does not always deliver potent vaccines to all parts of the world (Zaffran, 1996; Arya, 2001; Rexroad et al., 2002; Brandau et al., 2003). The vaccine stability problem is made more difficult by the complexity of certain vaccine antigens such as attenuated viruses or bacteria. These macromolecules are assemblies of various components (e.g., nucleic acids, proteins, lipids), and degradation in any of these components may adversely affect the potency of the entire vaccine. Vaccines in general are often lyophilized, or freeze-dried, to improve their storage stability, yet protective excipients must be chosen carefully to realize these improvements and there is a need to identify excipients that will provide commercially useful and physiologically acceptable preparations of alphavirus-based vaccines.
The Alphavirus genus includes a variety of viruses, all of which are members of the Togaviridae family. The alphaviruses include Eastern Equine Encephalitis Virus (EEE), Venezuelan Equine Encephalitis Virus (VEE), Everglades Virus, Mucambo Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEE), Sindbis Virus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus, O'nyong-nyong Virus, Ross River Virus, Barmah Forest Virus, Getah Virus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, Babanki Virus, Kyzylagach Virus, Highlands J Virus, Fort Morgan Virus, Ndumu Virus, and Buggy Creek Virus. The viral genome is a single-stranded, messenger-sense RNA, modified at the 5′-end with a methylated cap and at the 3′-end with a variable-length poly (A) tract. Structural subunits containing a single viral protein, capsid, associate with the RNA genome in an icosahedral nucleocapsid. In the virion, the capsid is surrounded by a lipid envelope covered with a regular array of transmembrane protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2. See Pedersen et al., J. Virol 14:740-744 (1974). The Sindbis and Semliki Forest viruses are considered the prototypical alphaviruses and have been studied extensively. See Schlesinger, The Togaviridae and Flaviviridae, Plenum Publishing Corp., New York (1986). The VEE virus has been studied extensively, see, e.g., U.S. Pat. No. 5,185,440.
The studies of these viruses have led to the development of techniques for vaccinating against the alphavirus diseases and against other diseases through the use of alphavirus vectors for the introduction of foreign genes. See U.S. Pat. No. 5,185,440 to Davis et al., and PCT Publication WO 92/10578. The use of alphavirus vectors to direct the expression of foreign genes in eukaryotes has become a topic of increasing interest. It is well known that live, attenuated viral vaccines are among the most successful means of controlling viral disease. However, for some virus pathogens, immunization with a live virus strain may be either impractical or unsafe. One alternative strategy is the insertion of sequences encoding immunizing antigens of such agents into a live, replicating strain of another virus. One such system utilizing a live VEE vector is described in U.S. Pat. Nos. 5,505,947 and 5,643,576 to Johnston et al. Another such system is described by Hahn et al., Proc. Natl. Acad. Sci. USA 89:2679-2683 (1992), wherein Sindbis virus constructs express a truncated form of the influenza hemagglutinin protein. Another system is the alphavirus replicon system, as described in U.S. Pat. No. 6,190,666 to Garoff et al., U.S. Pat. Nos. 5,792,462 and 6,156,558 to Johnston et al., U.S. Pat. Nos. 5,814,482, 5,843,723, 5,789,245, 6,015,694, 6,015,686 and 6,376,236 to Dubensky et al; U.S. Published Application No. 2002-0015945 A1 (Polo et al.), U.S. Published Application No. 2001-0016199 (Johnston et al.), Frolov et al. (1996) Proc. Natl. Acad. Sci. USA 93:11371-11377 and Pushko et al. (1997) Virology 239:389-401.
A new class of vaccines based on alphaviruses, a group of Togaviridae viruses has been developed, and thus, there is a need to identify alphavirus-based vaccine formulations that will provide commercially relevant stability during freezing, drying, storage, and rehydration. Alphavirus-based vaccines include live-attenuated alphavirus strains as well as alphavirus replicon particles (“ARP”, also designated VRP for virus-like replicon particles). VRP is also sometimes used herein as an acronym for a species of ARP known as VEE-based replicon particles. An ARP is a ˜70 nm propagation-defective virus-like particle which is produced in cells or cell cultures and incorporates a “replicon” that can express non-alphavirus genes within a virion shell comprising alphavirus structural proteins and membrane lipids. Thus, ARP and alphaviruses have very similar or identical surface compositions and are expected to behave similarly in formulation processes. In certain past studies, ARP formulations appeared to be relatively labile in solution, which has presented a hurdle in their commercial development.
Using an in vitro infectivity assay as an indicator of stability, it was established that freezing, drying, storage, and rehydration each presented unique stress vectors to ARP.